Fluorescent Reporter Libraries as Useful Tools for Optimizing Microbial Cell Factories: A Review of the Current Methods and Applications.

Delvigne F, Pêcheux H, Tarayre C - Front Bioeng Biotechnol (2015)

Bottom Line:
The level of protein/metabolite is thus proportional to a fluorescence signal.Actually, different kinds of reporter systems are available, as well as specific cultivation devices allowing the on-line recording of the fluorescent signal.Cell-to-cell variability is another important phenomenon that can be integrated into the screening procedures for the selection of more efficient microbial cell factories.

ABSTRACTThe use of genetically encoded fluorescent reporters allows speeding up the initial optimization steps of microbial bioprocesses. These reporters can be used for determining the expression level of a particular promoter, not only the synthesis of a specific protein but also the content of intracellular metabolites. The level of protein/metabolite is thus proportional to a fluorescence signal. By this way, mean expression profiles of protein/metabolites can be determined non-invasively at a high-throughput rate, allowing the rapid identification of the best producers. Actually, different kinds of reporter systems are available, as well as specific cultivation devices allowing the on-line recording of the fluorescent signal. Cell-to-cell variability is another important phenomenon that can be integrated into the screening procedures for the selection of more efficient microbial cell factories.

Figure 1: Diversity of experimental devices that can be used for investigating fluorescent reporter libraries. (*) Device not compatible with cell sorting. (**) Cell sorting is not considered in bioprocessing conditions.

Mentions:
Besides its importance in giving new insights in cell biology, e.g., for the analysis of the intrinsic and extrinsic component of phenotypic noise among microbial population (Swain et al., 2002), fluorescent reporters are also an important component for the development of new bioprocesses, i.e., for strain engineering and process optimization up to the large-scale production of bioproducts (Polizzi and Kontoravdi, 2014). From a fundamental perspective, fluorescent reporter libraries are available for several model organisms, including Escherichia coli K12 MG1655 (Zaslaver et al., 2006) and Saccharomyces cerevisiae (Newman et al., 2006). The two above-mentioned fluorescent reporter libraries have notably been used for the characterization of noise in protein expression (Newman et al., 2006; Silander et al., 2012). Indeed, molecular processes associated with DNA transcription and translation are subjected to different noise mechanisms leading a cell-to-cell variability in protein content among an isogenic microbial population (Sanchez et al., 2013). Clone libraries and experimental devices for the cultivation and the detection of fluorescent signal at the single-cell level have been specifically developed (Taniguchi et al., 2010). Besides these genome-scale libraries, fluorescent reporter system can also be used for the design of smaller libraries, e.g., for the estimation of the strength of several promoters that could be used for the expression of a protein of interest or for the design of synthetic metabolic pathways (Xu et al., 2012). This last application of fluorescent reporter is very important, since synthetic biology becomes widespread for the design of efficient cell factories, able to synthesize fuels and chemicals with high titer. Fluorescent proteins can also be found in more specific applications, such as the detection of the intracellular metabolite level (Schallmey et al., 2014) or the control of lab evolution (Reyes, 2012a,b), which will be detailed throughout this review. The exploitation of a fluorescent reporter library is greatly facilitated by the use of specific experimental devices. Indeed, the actual experimental toolbox dedicated to the use of fluorescent reporters allows for all the manipulations required in bioprocess optimization and scale-up and comprise specific cultivation devices, analytical tools, and clone selection tools (Figure 1). Among the cultivation device, a full range of culture volume is available, from micro- (picoliter) and mini-bioreactor (milliliter) to full-scale bioreactors (liter). Micro-bioreactors are based on microfluidic chips adapted to the culture of microorganisms. A nice example of micro-bioreactor has been developed by Grunberger et al. (2012, 2013), where a single microbial cell is isolated in a picoliter chamber perfused by fresh medium. The height of the picoliter chamber is designed in order to be slightly higher than the mean diameter of the microbial cells, so that microbial cells are maintained in the chamber and are continually fed with fresh medium whereas metabolites and by-products are continuously extracted. The perfusion mode of culture allows thus to cultivate microorganisms under constant environmental conditions. Imaging allows for the acquisition of the individual division rate and also the gene activity if linked with a fluorescent reporter system. A major limitation of the actual micro-bioreactor is that they are not designed to work in the operating modes generally met in industrial conditions, i.e., batch and fed-batch (Love, 2013; Grunberger et al., 2014). This limitation can be overcome by considering mini-bioreactors. This range of bioreactor involves the use of cultivation volume of around 1 ml (Klockner and Buchs, 2012). One of the most advanced mini-bioreactor platform to date is the Biolector system, and its extension Robolector (Funke et al., 2010). This device is based on a microplate and allows the parallel cultivation of 48 samples with on-line determination of biomass, pH, dissolved oxygen, and fluorescence. High oxygen transfer efficiency allows to carry out microbial culture in fully aerobic conditions and fed-batch and pH control are available, ensuring the compatibility of the results with those gained in conventional stirred bioreactor. Fluorescence sensor available in each well can be used to gain informations at the level of a fluorescent reporter system, but only at the bulk level. Other mini-bioreactor systems are now available; either based on the concept of “shaken” bioreactor (e.g., Micro 24-microreactor system developed by Pall) or “stirred” bioreactor (e.g., the 48-bioreactor system developed by 2mag) (Lattermann and Buchs, 2014). Single-cell results can be obtained by coupling the cultivation device to a robotic platform delivering the samples to a flow cytometer. Microbial phenotypic heterogeneity is a phenomenon that has gained a lot of attention, considering its potential impact on bioprocesses (Delvigne et al., 2014). Fluorescent reporter library is a technology of choice for investigating the effect of microbial phenotypic heterogeneity on bioprocesses and single-cell analytical devices compatible with bioreactor are needed at this level. Indeed, genome-scale investigation of GFP reporter libraries have led to a better understanding of the evolution of noise in gene and protein expression (Taniguchi et al., 2010; Silander et al., 2012) and the associated molecular mechanisms (Swain et al., 2002). Since on-line flow cytometry can be adapted to monitor phenotypic heterogeneity during a microbial culture by coupling a specific interface to the cultivation device (Brognaux et al., 2013), these mechanisms are now considered in bioprocessing conditions (Polizzi and Kontoravdi, 2014; Baert et al., 2015). Several interfaces have been built for this purpose and have been successfully used to monitor the activity of fluorescent reporter in bioprocess conditions (Abu-Absi et al., 2003; Arnoldini et al., 2013; Besmer et al., 2014; Delvigne et al., 2015). In the following section, the use of fluorescent reporter libraries will be illustrated for different biotechnological applications.

Figure 1: Diversity of experimental devices that can be used for investigating fluorescent reporter libraries. (*) Device not compatible with cell sorting. (**) Cell sorting is not considered in bioprocessing conditions.

Mentions:
Besides its importance in giving new insights in cell biology, e.g., for the analysis of the intrinsic and extrinsic component of phenotypic noise among microbial population (Swain et al., 2002), fluorescent reporters are also an important component for the development of new bioprocesses, i.e., for strain engineering and process optimization up to the large-scale production of bioproducts (Polizzi and Kontoravdi, 2014). From a fundamental perspective, fluorescent reporter libraries are available for several model organisms, including Escherichia coli K12 MG1655 (Zaslaver et al., 2006) and Saccharomyces cerevisiae (Newman et al., 2006). The two above-mentioned fluorescent reporter libraries have notably been used for the characterization of noise in protein expression (Newman et al., 2006; Silander et al., 2012). Indeed, molecular processes associated with DNA transcription and translation are subjected to different noise mechanisms leading a cell-to-cell variability in protein content among an isogenic microbial population (Sanchez et al., 2013). Clone libraries and experimental devices for the cultivation and the detection of fluorescent signal at the single-cell level have been specifically developed (Taniguchi et al., 2010). Besides these genome-scale libraries, fluorescent reporter system can also be used for the design of smaller libraries, e.g., for the estimation of the strength of several promoters that could be used for the expression of a protein of interest or for the design of synthetic metabolic pathways (Xu et al., 2012). This last application of fluorescent reporter is very important, since synthetic biology becomes widespread for the design of efficient cell factories, able to synthesize fuels and chemicals with high titer. Fluorescent proteins can also be found in more specific applications, such as the detection of the intracellular metabolite level (Schallmey et al., 2014) or the control of lab evolution (Reyes, 2012a,b), which will be detailed throughout this review. The exploitation of a fluorescent reporter library is greatly facilitated by the use of specific experimental devices. Indeed, the actual experimental toolbox dedicated to the use of fluorescent reporters allows for all the manipulations required in bioprocess optimization and scale-up and comprise specific cultivation devices, analytical tools, and clone selection tools (Figure 1). Among the cultivation device, a full range of culture volume is available, from micro- (picoliter) and mini-bioreactor (milliliter) to full-scale bioreactors (liter). Micro-bioreactors are based on microfluidic chips adapted to the culture of microorganisms. A nice example of micro-bioreactor has been developed by Grunberger et al. (2012, 2013), where a single microbial cell is isolated in a picoliter chamber perfused by fresh medium. The height of the picoliter chamber is designed in order to be slightly higher than the mean diameter of the microbial cells, so that microbial cells are maintained in the chamber and are continually fed with fresh medium whereas metabolites and by-products are continuously extracted. The perfusion mode of culture allows thus to cultivate microorganisms under constant environmental conditions. Imaging allows for the acquisition of the individual division rate and also the gene activity if linked with a fluorescent reporter system. A major limitation of the actual micro-bioreactor is that they are not designed to work in the operating modes generally met in industrial conditions, i.e., batch and fed-batch (Love, 2013; Grunberger et al., 2014). This limitation can be overcome by considering mini-bioreactors. This range of bioreactor involves the use of cultivation volume of around 1 ml (Klockner and Buchs, 2012). One of the most advanced mini-bioreactor platform to date is the Biolector system, and its extension Robolector (Funke et al., 2010). This device is based on a microplate and allows the parallel cultivation of 48 samples with on-line determination of biomass, pH, dissolved oxygen, and fluorescence. High oxygen transfer efficiency allows to carry out microbial culture in fully aerobic conditions and fed-batch and pH control are available, ensuring the compatibility of the results with those gained in conventional stirred bioreactor. Fluorescence sensor available in each well can be used to gain informations at the level of a fluorescent reporter system, but only at the bulk level. Other mini-bioreactor systems are now available; either based on the concept of “shaken” bioreactor (e.g., Micro 24-microreactor system developed by Pall) or “stirred” bioreactor (e.g., the 48-bioreactor system developed by 2mag) (Lattermann and Buchs, 2014). Single-cell results can be obtained by coupling the cultivation device to a robotic platform delivering the samples to a flow cytometer. Microbial phenotypic heterogeneity is a phenomenon that has gained a lot of attention, considering its potential impact on bioprocesses (Delvigne et al., 2014). Fluorescent reporter library is a technology of choice for investigating the effect of microbial phenotypic heterogeneity on bioprocesses and single-cell analytical devices compatible with bioreactor are needed at this level. Indeed, genome-scale investigation of GFP reporter libraries have led to a better understanding of the evolution of noise in gene and protein expression (Taniguchi et al., 2010; Silander et al., 2012) and the associated molecular mechanisms (Swain et al., 2002). Since on-line flow cytometry can be adapted to monitor phenotypic heterogeneity during a microbial culture by coupling a specific interface to the cultivation device (Brognaux et al., 2013), these mechanisms are now considered in bioprocessing conditions (Polizzi and Kontoravdi, 2014; Baert et al., 2015). Several interfaces have been built for this purpose and have been successfully used to monitor the activity of fluorescent reporter in bioprocess conditions (Abu-Absi et al., 2003; Arnoldini et al., 2013; Besmer et al., 2014; Delvigne et al., 2015). In the following section, the use of fluorescent reporter libraries will be illustrated for different biotechnological applications.

Bottom Line:
The level of protein/metabolite is thus proportional to a fluorescence signal.Actually, different kinds of reporter systems are available, as well as specific cultivation devices allowing the on-line recording of the fluorescent signal.Cell-to-cell variability is another important phenomenon that can be integrated into the screening procedures for the selection of more efficient microbial cell factories.

ABSTRACTThe use of genetically encoded fluorescent reporters allows speeding up the initial optimization steps of microbial bioprocesses. These reporters can be used for determining the expression level of a particular promoter, not only the synthesis of a specific protein but also the content of intracellular metabolites. The level of protein/metabolite is thus proportional to a fluorescence signal. By this way, mean expression profiles of protein/metabolites can be determined non-invasively at a high-throughput rate, allowing the rapid identification of the best producers. Actually, different kinds of reporter systems are available, as well as specific cultivation devices allowing the on-line recording of the fluorescent signal. Cell-to-cell variability is another important phenomenon that can be integrated into the screening procedures for the selection of more efficient microbial cell factories.